Methods for the immobilization of biologically active materials, especially enzymes, have undergone such rapid development in recent years that it is fair to say that support matrices and their preparation are rather mature fields of technology in, for example, enzyme catalyzed reactions of commercial importance. The impetus for their development initially was the conservation of enzymes; the use of enzymes in homogeneous reactions generally mandated the single use of enzymes. Because enzymes often are an expensive component of reactions, and sometimes the most expensive one, there arose the need to develop methods allowing multiple use of enzymes. Immobilization of enzymes on solid supports led to heterogeneous enzyme-catalyzed reactions where the immobilized enzymes could be readily removed, as in a stirred batch reactor, or could be employed in a continuous process, as in a fixed bed, but in either case permitted enzyme-catalyzed processes where the enzyme could be reused until its decreased activity made further use economically unfeasible.
Presently there are a variety of support matrices from which immobilized enzymes specifically, and immobilized biologically active materials generally, can be prepared. Some bind the enzyme, as exemplary of a biologically active material, via ionic interaction, others bind the enzyme via entrapment. In still others the biologically active material is immobilized by covalent bonding to the support or some intermediary linked to the support. Thus, the skilled worker has some realistic alternatives in his technological closet when seeking a support matrix with which to immobilize a biologically active substance.
A type of support matrix which has proved versatile and effective in a wide scope of operations is that described in U.S. Pat. No. 4,141,857. The particles of the matrix are incompressible, hard, unreactive materials, which are desirable properties for good flow characteristics in a packed bed reactor, composed chiefly of porous inorganic oxides coated with an inorganic resin. The latter results from the reaction of a polyamine with a large excess of a bifunctional organic reagent, especially a dialdehyde, to afford a crosslinked polyamine with a multiplicity of pendant aldehyde groups available for covalent bonding to enzymes. However versatile such a support matrix may be, in the course of some investigations we observed limitations which led to an investigation culminating in the present application.
Although enzymes necessarily are active in aqueous media, the substrates on which enzymes act span a large range of hydrophilic character. At one extreme of the spectrum are polyhydroxylic compounds such as sugars, such as glucose, which are highly polar, extremely hydrophilic materials. At or near the other end of the spectrum are triglycerides, especially those of long chain (C12+) fatty acids, where the long hydrocarbon tail of the fatty acids provide a highly hydrophobic environment. (Throughout this application the words "hydrophobic" and "lipophilic" will be used synonymously. Although interchangeable in the context of this application, the terms stress different aspects of the same physical property.) Even though enzymes within a cell are in an aqueous medium, nonetheless the microenvironment of enzymatic reactions is not homogeneous and will in part reflect the hydrophilic-hydrophobic character of the substrate being acted upon. One can expect the microenvironment within which a sugar is enzymatically isomerized will be highly hydrophilic. One also can expect a substantially different microenvironment for the hydrolysis of triglycerides; the long hydrocarbon tail of fatty acids is quite lipophilic, whereas the reaction site of the ester moiety in combination with the reactant water molecule is rather hydrophilic.
A natural consequence of the varying hydrophilic-hydrophobic properties of enzyme substrates is that the particular enzyme acting on a substrate also can be expected to reflect a varying balance of hydrophilic-hydrophobic properties. Although enzymes generally can be expected to be amphiphilic (that is, exhibiting both hydrophilic and hydrophobic properties), nonetheless enzymes acting on hydrophobic substrates can be expected to have more hydrophobic character than enzymes acting on hydrophilic substrates. When an enzyme is removed from its natural environment and immobilized on a support matrix, additional factors must be considered for optimization of the enzyme-mediated reaction in question. In this regard the example of triglyceride hydrolysis is instructive. Triglycerides are water insoluble, and the hydrolytic medium usually is a highly hydrophobic triglyceride phase in which is dispersed a small amount of water. Reaction probably occurs at the oil-water interface, with its attendant discontinuity in hydrophilic-hydrophobic properties. The enzymes catalyzing triglyceride hydrolysis, which collectively are known as lipases, probably have hydrophobic character in most regions, although the active site is likely to be more hydrophilic. At the same time it is desirable that the support matrix have regions of hydrophilicity to accommodate the reactant water molecule and to gain access to the water-oil interface. The result that emerges is that an effective immobilized lipase is one where the support matrix is amphiphilic, with hydrophobic regions for binding the enzyme and hydrophilic regions for binding of water.
The foregoing conceptualization, though useful, has limitations. It appears that, for example, not all lipases are comparably hydrophobic. For some lipases, presumably those with the greatest hydrophobic character, immobilization on a support with hydrophobic properties effectively binds the enzyme without interfering with its expression of enzyme activity and the resulting immobilized enzyme manifests a higher activity than one immobilized on a more hydrophilic support. Other lipases appear to be less hydrophobic, and for these immobilization on a hydrophobic support does not result in any increase of enzymatic activity, although no decrease in activity may be observed relative to the enzyme immobilized on a hydrophilic support.
There is another aspect of lipases in the hydrolysis of fats and oils which my invention addresses. The enzymatic hydrolysis of lipids using a 1,2,3-nonspecific lipase often is slow with complete hydrolysis difficult to achieve. During the hydrolysis, a large amount of diglycerides are accumulated in the reaction mixture because lipases hydrolyze diglycerides slowly compared to the hydrolysis of triglycerides. To avoid the accumulation of diglycerides in the reaction mixture, an excess amount of lipase is used to carry out a successful hydrolysis. The use of an immobilized lipase requires an enzyme support which is able to absorb a large amount of lipase and to express the full catalytic activity of the absorbed enzyme. The support matrix of this invention and the immobilized lipases prepared therefrom are particularly effective in these regards. In addition, the support has both polar and hydrophobic sites so as to behave as an emulsifier between oil and water.
Our invention is a support matrix for the immobilization of biologically active material having both a hydrophobic region for binding biologically active material, especially with substantial hydrophobic character, and a hydrophilic region for binding with one or more components of the reaction medium. Such a support matrix has the advantages of expressing higher activity of lipophilic enzymes, such as lipases, while affording access to hydrophilic sites, especially to the oil-water interface at which an enzyme-catalyzed reaction occurs.